The field of the present invention is in the area of immunotherapy, especially as it relates to the treatment and/or prophylaxis of cancer and/or tumors and compositions therefore.
The antitumor response is a very effective mechanism for eliminating tumors from the body. T cells play a major role in this elimination. To activate tumor-specific T cells, an initial signal need to be delivered by a T cell receptor interacting with the antigen-major histocompatibility complex (MHC) complexes expressed on antigen presenting cells (APC) (FIG. 1). The second signal is provided by costimulatory cell adhesion molecules (CoCAMS) present on the T cells interacting with their counter-receptors expressed on the APCs [Norton et al. (1992) J. Immunol. 149:1556-1561; Linsley et al. (1991) J. Exp. Med. 173:721-730; Azuma et al. (1992) J. Exp. Med. 175:353-360; June et al. (1994) Immunol. Today 15:321-331; Galvin et al. (1992) J. Immunol. 149:3802-3808]. When both signals are delivered, a normal T cell immune response occurs. The T cells expand, proliferate and develop into antigen-specific cytotoxic T lymphocytes (CTLs).
Tumor cells may also function as APC. These tumor cells, expressing an MHC -antigen complex and costimulatory adhesion molecules, can provide the necessary signals for the generation of tumor-specific CTLs. (FIG. 1). However, in the absence of the second signal from the tumor cells, tumor-specific T cells die [Tan et al. (1993) J. Exp. Med. 177:165-173; Gimmi et al. (1993) Proc. Natl. Acad. Sci. USA 90:6586-6590; Harding et al. (1992) Nature 356:607]. Tumors, by down-modulating cell adhesion molecules, can escape immune attack. Thus, by not providing the second signal the tumors not only avoid the immune system but also can, in effect, kill the T cells that are specific for the tumors [Townsend et al. (1993) Science 259:368-370; Li et al. (1994) J. Immunol. 153:421-428; Chen et al. (1994) J. Exp. Med. 179:532xe2x80x94532; Chen et al. (1992) Cell 71:1092-1102].
There are many CoCAMs important in development of the tumor-specific immune response. The most important receptor-ligand pairs in the immune system are interactions between CD2 and LFA-3, CD11 and ICAM-1, and CD28 with B-7.1 or B-7.2 [Johnson and Jenkins (1993) Immunol. Res. 12:48-64; Chen et al. (1993) Immunol. Today 14:483-486; Schwartz, R. H. (1992) Cell 1065-1068; Lanier, L. (1993) Ann. NY Acad. Sci. 677:86-93]. ICAMs are intercellular adhesion molecules. Among these three pairs, the CD28/CB7 interaction appears to provide the most important second signal for tumor-specific immunity [Townsend et al. (1993) supra; Li et al. (1994) supra; Chen et al. (1994) supra; Chen et al. (1992) supra].
Recently, CoCAMs, such as B7-1 and ICAM-1 [Chen et al. (1993) J. Immunol. 151:244-255], were expressed on tumor cells by gene transfer. These modified tumor cells did not grow in syngeneic mice, and also induced immunity against the parental tumor cell. This costimulation provided by the tumor cell is only necessary at the onset of the immune response. Once this is initiated, CTLs do not need CD28/B7 interaction to kill the target cell. Therefore, one can modify a tumor cell with B7-1 and stimulate the immune system to attack the unmodified tumor cell. Thus, modified tumor cells can be used as cancer vaccines.
Recently, Guo et al. [Guo et al. (1994) Science 265, 518-520] used another approach to prepare tumor vaccines. They generated tumor hybridomas by fusing hepatoma cells with activated B cells. These hybridomas expressed the tumor antigens as well as all the CoCAMs from the B cell. This hybridoma was able to immunize mice against the hepatoma.
There is a longfelt need in the art for compositions and methods effective in neoplasia, tumor and/or cancer prophylaxis and immunotherapy and a need for methods for making same without the introduction of cells carrying recombinant DNA into the vaccine preparations. The alternative methodology which the present invention uses is protein transfer.
It is an object of the present invention to provide compositions useful in immunotherapy, especially for the prophylaxis or treatment of neoplastic conditions and/or tumors, including cancer, for example, those tumors or cancers in which the cell membranes lack the CoCAM surface molecules including, but not limited to the B7-1 protein. The B7-1 protein is involved in costimulation of cells of the immune system via interaction with the CD28 protein of T cells.
Specifically exemplified compositions include those which comprise a fusion protein consisting of the extracellular, CD28 binding domain of B7-1 in combination with a glycosylated phosphatidylinositol (GPI) anchor domain, which GPI anchor domain is from membrane proteins including, but not limited to, decay accelerating factor, CD16, CD16B, and (preferably) LFA-3. As used herein, B7-1 is used synonymously with CD80. A specifically exemplified fusion protein consists of amino acids 1-243 of the B7-1 protein covalently joined to amino acids 193-234 of CD16B (See Freeman et al. (1989) infra, incorporated by reference herein). Other CoCAMs which can be modified to contain a GPI anchor include B7.2, ICAM-1, ICAM-2, VCAM-1 and LFA-3.
It is a further object of the invention to provide methods for incorporating the fusion protein(s) of the present invention into tumor cell membranes which in nature lack a CoCAM protein, such as the B-7.1 protein. The naturally occurring neoplastic cells or naturally occurring neoplastic cell membranes are prepared using art-known techniques, and then the fusion protein is added under conditions which allow the fusion protein to become embedded in the tumor cell membranes via a GPI anchor portion.
It is a further object of the invention to provide immunotherapeutic compositions comprising the neoplastic cells or neoplastic cell membranes (as isolated membranes or as intact or irradiated cells) into which the GPI-CoCAM fusion protein of the present invention has been embedded in combination with a pharmaceutically acceptable vehicle. Optionally, the immunotherapeutic composition can further include an immunological adjuvant which will enhance the cytotoxic immune response of an animal, including, but not limited to, a human, to which the composition is administered. Administration of the immunotherapeutic composition occurs by a route suitable for stimulating an immune response, and in particular, for activating a cellular immune response. Alternatively, the B7-1/CD16 fusion protein can be incorporated within the tumor cell membranes before the step of membrane purification (the fusion protein can be introduced into intact tumor cells) and preferably after homogenization of the tumor tissue to maximize cell membrane surface area available for embedding of B7-1/GPI fusion protein. Optionally, the immunotherapeutic compositions of the present invention can further include additional GPI-anchored fusion proteins which augment the costimulatory effect of the anchored B7-1 or other CoCAM protein. Further or in the alternative, these compositions of the present invention can additionally include at least one cytokine in an amount effective for the augmentation of the cytotoxic immune response. For this purpose, particularly preferred cytokines include, without limitation, interleukin-12 (IL-12) and interleukin-6 (IL-6).
Within the scope of the present invention are methods for the treatment of tumors and certain other neoplastic conditions, where these methods include the step of administering the immunotherapeutic composition comprising the B7-1/GPI or other GPI-CoCAM fusion protein, preferably the composition further includes an antigen suitable for stimulating cellular immunity and activating a cytotoxic response, as is well understood in the art, to an animal, including but not limited to, a human, where that animal has or has had at least one tumor having at least one marker surface antigen in common with those tumor cells used in the preparation of the immunotherapeutic composition of the present invention and where that tumor naturally lacks a surface CoCAM, as specifically exemplified for B7-1. The present invention also encompasses prophylactic methods for prevention of tumors or cancers, the method comprising the step of administering an immunogenic composition containing the tumor cell membrane (or tumor cell) preparation into which the GPI-anchored B7-1 fusion protein has been incorporated, in an amount effective for generating a cytotoxic immune response specific for the tumor or cancer cell. Optional additional ingredients in the immunotherapeutic composition include, without limitation, additional GPI-anchored costimulatory cell surface proteins and/or cytokines, e.g., IL-12 and optionally, IL-6.